RESUMEN
Abdominal aortic aneurysm (AAA) is a complex disorder that has a significant impact on the aging population. While both genetic and environmental risk factors have been implicated in AAA formation, the precise genetic markers involved and the factors influencing their expression remain an area of ongoing investigation. DNA methylation has been previously used to study gene silencing in other inflammatory disorders and since AAA has an extensive inflammatory component, we sought to examine the genome-wide DNA methylation profiles in mononuclear blood cells of AAA cases and matched non-AAA controls. To this end, we collected blood samples and isolated mononuclear cells for DNA and RNA extraction from four all male groups: AAA smokers (n = 11), AAA non-smokers (n = 9), control smokers (n = 10) and control non-smokers (n = 11). Methylation data were obtained using the Illumina 450k Human Methylation Bead Chip and analyzed using the R language and multiple Bioconductor packages. Principal component analysis and linear analysis of CpG island subsets identified four regions with significant differences in methylation with respect to AAA: kelch-like family member 35 (KLHL35), calponin 2 (CNN2), serpin peptidase inhibitor clade B (ovalbumin) member 9 (SERPINB9), and adenylate cyclase 10 pseudogene 1 (ADCY10P1). Follow-up studies included RT-PCR and immunostaining for CNN2 and SERPINB9. These findings are novel and suggest DNA methylation may play a role in AAA pathobiology.
Asunto(s)
Aneurisma de la Aorta Abdominal/patología , Metilación de ADN , Anciano , Anciano de 80 o más Años , Aorta/metabolismo , Aorta/patología , Aneurisma de la Aorta Abdominal/genética , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Islas de CpG , ADN/aislamiento & purificación , ADN/metabolismo , Humanos , Inmunohistoquímica , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Masculino , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Seudogenes/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Serpinas/genética , Serpinas/metabolismo , Fumar , CalponinasRESUMEN
BACKGROUND: Abdominal aortic aneurysm (AAA) is a dilatation of the aorta affecting most frequently elderly men. Histologically AAAs are characterized by inflammation, vascular smooth muscle cell apoptosis, and extracellular matrix degradation. The mechanisms of AAA formation, progression, and rupture are currently poorly understood. A previous mRNA expression study revealed a large number of differentially expressed genes between AAA and non-aneurysmal control aortas. MicroRNAs (miRNAs), small non-coding RNAs that are post-transcriptional regulators of gene expression, could provide a mechanism for the differential expression of genes in AAA. METHODS: To determine differences in miRNA levels between AAA (n = 5) and control (n = 5) infrarenal aortic tissues, a microarray study was carried out. Results were adjusted using Benjamini-Hochberg correction (adjusted p < 0.05). Real-time quantitative RT-PCR (qRT-PCR) assays with an independent set of 36 AAA and seven control tissues were used for validation. Potential gene targets were retrieved from miRNA target prediction databases Pictar, TargetScan, and MiRTarget2. Networks from the target gene set were generated and examined using the network analysis programs, CytoScape® and Ingenuity Pathway Core Analysis®. RESULTS: A microarray study identified eight miRNAs with significantly different expression levels between AAA and controls (adjusted p < 0.05). Real-time qRT-PCR assays validated the findings for five of the eight miRNAs. A total of 222 predicted miRNA target genes known to be differentially expressed in AAA based on a prior mRNA microarray study were identified. Bioinformatic analyses revealed that several target genes are involved in apoptosis and activation of T cells. CONCLUSIONS: Our genome-wide approach revealed several differentially expressed miRNAs in human AAA tissue suggesting that miRNAs play a role in AAA pathogenesis.